Mycobacterium tuberculosis (MTB) infects one-third of the world population and is responsible for 2-3 million deaths each year. Eight million people develop clinical disease each year. A protective vaccine is not yet available and multi-drug resistant MTB-strains pose a threat in developing and industrialized countries. Multiple defense mechanisms are available to contain the intracellular MTB infection to latency, including antigen reactive γδ+ T-cells, CD4+ T-cells which may differentiate into memory T-lymphocytes and CD8+ T-cells instrumental in maintaining anti-MTB control.
Latent MTB infection represents a major threat both for the infected individual and for spreading the infectious agent to other individuals. Weakened cellular immunity associated with age, malnutrition, immunosuppression, or coinfection with HIV leads to loss of MTB containment. Disease containment in TB involves a complex network of immune cells and their ability to mount and maintain effective immune responses. Our knowledge of all engaged immune events involved in maintaining latency of this disease in humans is yet to be complete. The importance of a coordinated cellular immunity to combat this intracellular pathogen is however indisputable. The essential role of MHC class II-restricted CD4+ as well as MHC class I-restricted CD8+ T-cells is well established (recently reviewed in). One of the major weapons of these T-cells is the production of cytokines, e.g. IFN-γ and TNF-α, that can activate macrophages and induce their production of bacteriocidal molecules. Nonclassically restricted CD8+ lymphocytes, CD4−CD8− and CD1-restricted T-cells, γδ cells and NK cells have also been reported to recognize mycobacterial lipid antigens presented on CD1 molecules and to be involved in disease control.
This knowledge is derived largely from studies in mice. Most studies on latent or active human TB or exposure to TB to date are limited to the analysis of peripheral blood lymphocytes (PBLs) and do not address the immunological situation at the primary site of MTB infection, the lung. The investigation of latent TB is, moreover, hampered for lack of an appropriate animal model for this state of disease.
The tine-skin test (TST) assesses the presence of cellular immune reactivity directed against mycobacterium species as a response to intradermal PPD injection. It can not differentiate between infection with mycobacteria other than tuberculosis (MOTT), vaccination with Bacille Calmette Guerin (BCG) and clinically relevant M. tuberculosis infection. It also provides no information about the exact antigen-specificity and involvement of CD4 or CD8+ T-cells and their differentiation status or homing capacity. Several variables are important to determine effective containment of mycobacterium species: CD4+ T-cells appear to be crucial to fight off infection in acute infection, CD8+ T-cells are more likely to be involved in maintaining long-term anti-MTB immune responses. Also, different T-cell subsets are important to maintain a strong and long-lived immune response. A pool of ‘precursor’ T-cells, immediate acting ‘effector’ T-cells, or memory T-lymphocytes, which are free to leave the bloodstream and to enter the site of infection with different potentials of proliferation or cytokine production, may be advantageous in order to achieve maximum and long-lived protection. A precise dissection of immune responses in different target antigens has heretofore not been known, but would allow discrimination between patients with MTB, MOTT or M. leprae infection. Exclusively ESAT-6 is expressed in MTB (and M. leprae) but not in BCG, or MOTT which designates this protein as an attractive vaccine candidate and target to gauge bona fide MTB-specific T-cell responses. Conversely, HLA-DR4 restricted Ag85b epitopes may—in addition to MTB diagnostics—be useful to enumerate T-cells specific for M. ulcerans, the causative agent of Buruli ulcer or M. avium intracellulare, a frequent cause for infections in immunocompromised patients. Thus, the TST as well as the IFNγ response assay (IGRA) are not able to address the above mentioned facets of an MTB-specific and effective immune response, since both assay systems implement PPD (purified protein derivative), a mix of different target proteins and lipoproteins as the stimulating agents and do not implement defined antigen specific peptide epitopes. A similar situation is true for the ELISPOT assay, which requires in most cases a 72 hr incubation period and does not allow simultaneous T-cell enumeration and T-cell marker analysis associated with T-cell homing and differentiation.
A more recently developed method of detecting antigen-specific T cells utilizing tetramers, oligomers or multimers of major histocompatibility complex (MHC) molecules has revolutionized T cell analysis. For example, MHC tetramer complexes are formed by the association of four MHC monomers, for example, four MHC class I molecule/β2-microglobulin monomers, with a specific peptide antigen and a detectable label such as a fluorochrome. Such MHC class I molecule tetramer complexes bind to a distinct set of T cell receptors on a subset of CD8+ T cells, including cytotoxic T lymphocytes (CTLs). CTLs, which are effector CD8+ T cells, do not necessarily represent the whole antigen-specific pool of CD8+ T cells. In this respect, the LDA and cytokine assay both detect CTLs or subpopulations of CTLs, whereas the MHC tetramer method can detect all antigen-specific CD8+ T cells, including naive and anergic CD8+ T cells, which do not exhibit effector functions. By mixing the MHC tetramers with peripheral blood lymphocytes or whole blood, and using flow cytometry as a detection system, a count of all T cells that are specific for a peptide and its matched allele is provided. As such, the MHC tetramers allow for the measurement of a cellular response against a specific peptide.
Spectratyping of the TCR repertoire allows for each individual TCR variable CDR3 profile obtained from 24 TCR VB families to be depicted as a function of the CDR3 length. Each peak represents 3 bp coding for one amino acid residue, with 9 or 10 amino acids being identified in each CDR3 profile. In the three-dimensional spectratype, the area of the entire CDR3 analysis is estimated as 100% for each TCR VB family, and the area under the curve for each individual CDR3 peak is expressed as the percentage of the entire CDR3 area, Spectratyping reflects the structural composition of the TCR repertoire, but does not provide information pertaining to the quantity of each TCR VA plus VB family, since the PCR-based amplification of the variable regions is not quantitative.
The use of MHC tetramers to analyze T cell specificity is quantitative; it does not require the use of radioactive dyes; and it is readily adapted to high throughput assay formats. In addition, the method can be performed quickly and, therefore, can be used to examine fresh blood or tissue samples. Where the MHC tetramer complex includes a fluorescent label, a cell population including T cells can be further stained with one or more other fluorescently labeled molecules, for example, fluorescently labeled molecules specific for other cell surface molecules and analyzed using flow cytometry, thus allowing additional characterization of the responding cells. In this case, the additional fluorescent label is selected to fluoresce at a wavelength that is readily distinguishable from the label(s) used to stain the target cells. Furthermore, MHC tetramer analysis is not toxic to the labeled cells and, therefore, tetramer binding cells can be sorted into uniform populations by flow cytometry and examined by additional assays to confirm their functional ability, for example, the ability to proliferate in response to antigen.
Therefore, there is a need in the art for methods of diagnosing in humans the factors responsible for containing MTB and sustaining latency in the human lung. The ex vivo identification of anti-MTB reactive CD4+ or CD8+ T-cells is desirable in order to provide biologically meaningful surrogate markers for gauging the efficacy of novel vaccine candidates, for diagnosing and monitoring latent MTB infections, for detecting MTB exposure, for testing for eradication of MTB in the course of already established and novel treatment strategies, and for defining the immunological threshold required to maintain ‘protective immunity’.